Geminiviruses threaten food security and agriculture, infecting key crop species, especially in tropical and subtropical regions (Gilbertson et al., Ann. Rev. Virol 0.2, 67-93 (2015)). Geminiviruses are characterized by their twin icosahedral capsids and small, single-stranded DNA (ssDNA) genome (approximately 2.7 kb) (Hanley-Bowdoin et al., Nat. Revs. Microbiol. 11, 777-788 (2013)). A study examining genome-wide pairwise sequence identity, genome organization, host range, and insect transmission vector recently classified the family Geminiviridae into seven genera: Begomovirus, Curtovirus, Topocuvirus, Mastrevirus, Becurtovirus, Turncurtovirus, and Eragroviru (Varsani et al., Arch. Virol. 1-11 (2014)). Begomoviruses infect dicotyledonous plants via the silverleaf whitefly (Bemisia tabaci) vector. The genomes of Begomoviruses are composed of one (A, monopartite) or two (A and B, bipartite) components. The A and B components of the virus share a common region with nearly identical nucleotide sequences (Fondong, V. N. Mol. Plant Pathol. 14, 635-649 (2013)).
Effective strategies for controlling geminiviruses remain expensive and inefficient due to mixed virus infections and the patho-interaction of vectors, viruses, and host plants (Gilbertson et al., Development of Integrated Pest Management (IPM) Strategies for Whitefly (Bemisia tabaci)-Transmissible Geminiviruses. 323-356 (2011)). However, recent work showed that site-specific nucleases can directly target and cleave the viral genome. This cleavage of the viral genome leads to the generation of double strand breaks (DSBs), which are either repaired by the imprecise non-homologous end-joining repair (NHEJ) machinery or by precise homology-directed repair (HDR) (Ebina et al., Sci. Rep. 3, 2510 (2013); Ali et al., Genome Biol. 16, 1-11 (2015); Aouida et al., Curr. Genet. 60, 61-74 (2014)). The presence of unrepaired DSBs ultimately leads to degradation of the virus molecules (Zaidi et al., Trends Plant Sci. 21, 279-281 (2016)). Virus variants generated by NHEJ can replicate and move systemically only if NHEJ maintains the proper “frame” for translation and does not compromise protein function. Several site-specific nuclease platforms have been developed with potential applications for targeted interference against viral genomes.
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) is an adaptive molecular immunity system used by bacteria and Archaea to fend off invading phages and conjugative plasmids (Wright et al., Cell 164, 29-44 (2016); Hsu et al., Cell 157, 1262-1278 (2014); Barrangou et al., Science 315, 1709-1712 (2007)). The CRISPR/Cas9 system has been harnessed for targeted mutagenesis and genome editing of eukaryotic genomes, including plants (Liu et al., Curr. Opinion Plant Biol. 30, 70-77 (2016); Nekrasov et al., Nat. Biotech. 31, 691-693 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013); Ali et al., Mol. Plant 8, 1288-1291 (2015)).
The CRISPR/Cas9 machinery is composed of Cas9 (a site-specific DNA endonuclease) and a synthetic single guide RNA (sgRNA, alternatively designated gRNA). The sgRNA, which carries 20-nuclecotides of target sequence information, is used to direct the Cas9 endonuclease to its genomic target sequence, which must precede a tri-nucleotide sequence known as the protospacer-associated motif (PAM). Streptomyces pyogenes Cas9 recognizes the PAM sequence NGG and cleaves three nucleotides preceding this PAM sequence on complementary and non-complementary strands (Wright et al., Cell 164, 29-44 (2016); Hsu et al., Cell 157, 1262-1278 (2014); Barrangou et al., Science 315, 1709-1712 (2007); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013)). Cas9 variants with improved specificity have recently been characterized (Zetsche et al., Cell 163, 759-771 (2015); Slaymaker et al., Science 351, 84-88 (2016); Davis et al., Nat. Chem. Biol. 11, 316-318 (2015); Kleinstiver et al., Nature 523, 481-485 (2015)).
It the portability of the CRISPR/Cas9 machinery to confer molecular immunity against eukaryotic viruses, including plant DNA viruses (Karimova et al., Sci. Rep. 5, 13734 (2015); Lin et al., Mol. Ther. Nucl. Acids (2014); Ramanan et al., Sci. Reports 5, 10833 (2015); Hu et al., Proc. Natl. Acad. Sci. U.S.A. 111, 11461-11466 (2014); Chaparro-Garcia et al., Genome Biol 16, 254 (2015)). CRISPR/Cas9 machinery can target coding and non-coding sequences of different geminiviruses (Baltes et al., Nat. Plants 1, 15145 (2015); Ji et al., Nat. Plants 1, 15144 (2015)). This targeting results in reduced viral accumulation and delayed or abolished symptoms. However, different sgRNAs have different targeting efficiencies for coding or noncoding sequences. Numerous reports describe the emergence of geminiviruses with altered pathogenicity and subsequent changes in the severity of disease symptoms in infected plants, resulting from recombination-mediated genetic changes or reassortment among different viral genomes. Targeting the viral genome opens up various possibilities, including degradation and/or repair of these genomes. Under natural field conditions, where mixed viral infections exist, targeting a single virus, generating DSBs, and initiating cellular repair could induce recombination or generation of viral variants capable of replication and survival. Since the nature of the target viruses largely determines the efficiency of interference by the CRISPR/Cas9 system, production of durable resistance requires the establishment of criteria for selecting which virus sequences to target.
It has proven difficult and expensive to control or manage the disease caused by the Tomato Yellow Leaf Curl Virus (TYLCV). Several approaches for disease resistance focus on insecticide treatments of the viral transmission vector whitefly (Bemicia tabaci) (Lapidot et al., (2014) Advances Virus Res. 90: 147-206). Breeding for resistance is challenging due to the linkage of genes of poor fruit quality to the resistance locus. Several attempts have been made to engineer tomato for resistance to TYLCV including the over-expression of the viral proteins CP, C4, or the IR noncoding sequences (Yang et al., (2004) Phytopathol. 94: 490-496). It has been shown that binding to the origin of replication by the replication protein (Rep) interferes with the viral replication and lead to viral resistance. Synthetic zinc finger protein has been used to block the Rep protein of the beet severe curly top virus (BSCTV) from binding to the origin of replication resulting in virus resistance (Sera T. (2005) J. Virol. 79: 2614-2619). Moreover, such technology was applied to TYLCV through the interference with the Rep protein binding to the origin of replication (Koshino-Kimura et al., (2009) Nucleic Acids Symp. Series 53: 281-282; Mori et al., (2013) Mol. Biotech. 54: 198-203). Nevertheless, effective means to control or manage the TYLCV disease has proven challenging. Therefore, developing effective technologies for viral resistance is needed to increase the yield of crop species (Galvez et al., (2014) Plant Sci. 228: 11-25)).